The long-term goal of the proposed research project is to understand the molecular mechanisms of cellular responses to a variety of stresses in mammalian cells. During the next granting period, we will investigate the effect of metabolic oxidative stress on TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptotic death. We postulate that tumor microenvironment, in particular glucose deprivation, enhances TRAIL-induced cytotoxicity by facilitating the mitochondria-dependent pathway and/or -independent pathway. Low glucose concentrations induce metabolic oxidative stress and subsequently promote cytochrome c release in the presence of TRAIL. Cytochrome c release promotes the mitochondria-mediated caspase signal transduction pathway. We also hypothesize that metabolic oxidative stress-induces an elevation of ceramide which activates ceramide-activated protein phosphatase (CAPP) and/or inactivates phosphoinositide-3 kinase [PI(3)K]. The activation of CAPP and/or inactivation of PI(3)K inactivates Akt through dephosphorylation and consequently down-regulates the expression of FLIP, an antiapoptotic protein, by inhibiting NF-kappaB signal transduction pathway. The specific aims of this project are to examine (1) the effect of low glucose concentrations on TRAIL-induced cytotoxicity, (2) the effect of low glucose concentrations on the TRAIL-activated apoptotic pathway, (3) cross-talk between the mitochondria-dependent caspase pathway and the apoptosis associated protein-regulated caspase pathway, (4) the role of the metabolic oxidative stress-ceramide-PI(3)K/CAPP-Akt-NF-kappaB-FLIP pathway in TRAIL sensitivity, (5) the effect of a variety of other characteristic features of the tumor microenvironment on TRAIL cytotoxicity. The proposed studies (Aim 1) will use survival determination assays to examine TRAIL-induced cytotoxicity in various concentrations of glucose. The second aim will focus on the use of biochemical approaches to investigate how low glucose concentrations promote the TRAIL-activated caspase signal transduction pathway. The studies for Aim 3 will use molecular and biochemical approaches to elucidate cross-talk between the mitochondria-dependent caspase pathway and apoptosis associated protein-regulated caspase pathway. We will employ molecular genetics and biochemical techniques to elucidate the role of the PI(3)K/CAPP-Akt-FLIP pathway in the low glucose-enhanced TRAIL cytotoxicity (Aim 4). Finally we will integrate different aspects of the signaling pathways. We believe that the successful outcome of this study will support the development and clinical application of TRAIL for the treatment of human cancer.